In order to define the origin of DNA replication in Drosophila mitochondria, mitochondrial DNA treated with restriction enzymes has been cloned in Escherichia coli. Using cloned plasmid DNA containing the DNA replication origin, we currently have been determining the nucleotide sequence. At the same time, an in vitro DNA replication system which resembles the in vivo system has been developed using native mitochondrial DNA and the chimeric DNA.